EPSP synthase highly tolerant to glyphosate

ABSTRACT

An isolated polypeptide comprising an amino acid sequence that is at least 62% identical to SEQ ID NO:2. The polypeptide, when present in a cell, increases the cell&#39;s ability to tolerate glyphosate. Also disclosed are related nucleic acid, antibody, vector, transgenic plant, as well as uses thereof.

RELATED APPLICATION

The present application claims priority to PCT/CN03/00651, filed on Aug.8, 2003, entitled “EPSP SYNTHASE HIGHLY TOLERANT TO GLYPHOSATE AND ITSCODING SEQUENCE”, which is hereby incorporated by reference as if fullyset forth herein.

FIELD OF THE INVENTION

The present invention generally relates to5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. More specifically,the invention relates to an EPSP synthase gene encoding a polypeptidehighly tolerant to glyphosate, and products containing the gene.

BACKGROUND OF THE INVENTION

Glyphosate is a non-selective, broad spectrum and post-emergenceherbicide. Glyphosate applied to foliage is absorbed by leaves andrapidly moves through the plant. Once absorbed, it prevents the plantfrom producing essential aromatic amino acids. This reduces theproduction of protein in the plant, and inhibits plant growth.

EPSP synthase is the sixth enzyme on the shikimate pathway, which isessential for the synthesis of aromatic amino acids. Based on thestudies of chemically induced aroA mutants, it has been confirmed thatEPSP synthase is encoded by the aroA gene. Recent advances in geneticengineering have made it possible to produce transgenic plants withunique characteristics of agronomic importance. Generation of glyphosatetolerant transgenic plants (e.g., containing glyphosate tolerant EPSPsynthase) will reduce the cost for weed control.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a novel EPSP synthase that ishighly tolerate to glyphosate. The amino acid sequence (SEQ ID NO:2) ofthe newly discovered EPSP synthase and its coding nucleic acid sequence(SEQ ID NO:1) are shown below:

ctcctacagt tagggcaagt cccccaccac tcgacaagc atg gcg tgt ttg cct 54                                           Met Ala Cys Leu Pro 5 gat gattcg ggt ccg cat gtc ggc cac tcc acg cca cct cgc ctt gac 102 Asp Asp SerGly Pro His Val Gly His Ser Thr Pro Pro Arg Leu Asp 21 cag gag cct tgtacc ttg agt tcg cag aaa acc gtg acc gtt aca ccg 150 Gln Glu Pro Cys ThrLeu Ser Ser Gln Lys Thr Val Thr Val Thr Pro 37 ccc aac ttc ccc ctc actggc aag gtc gcg ccc ccc ggc tcc aaa tcc 198 Pro Asn Phe Pro Leu Thr GlyLys Val Ala Pro Pro Gly Ser Lys Ser 53 att acc aac cgt gcg ctg ttg ctggcg gca ttg gcc aag ggc acc agc 246 Ile Thr Asn Arg Ala Leu Leu Leu AlaAla Leu Ala Lys Gly Thr Ser 69 cgt ttg agc ggt gcg ctc aaa agc gat gacacg cgc cac atg tcg gtc 294 Arg Leu Ser Gly Ala Leu Lys Ser Asp Asp ThrArg His Met Ser Val 85 gcc ctg cgg cag atg ggc gtc acc atc gac gag ccggac gac acc acc 342 Ala Leu Arg Gln Met Gly Val Thr Ile Asp Glu Pro AspAsp Thr Thr 101 ttt gtg gtc acc agc caa ggc tcg ctg caa ttg ccg gcc cagccg ttg 390 Phe Val Val Thr Ser Gln Gly Ser Leu Gln Leu Pro Ala Gln ProLeu 117 ttc ctc ggc aac gct ggc acc gcc atg cgc ttt ctc acg gct gcc gtg438 Phe Leu Gly Asn Ala Gly Thr Ala Met Arg Phe Leu Thr Ala Ala Val 133gcc acc gtg caa ggc acc gtg gta ctg gac ggc gac gag tac atg caa 486 AlaThr Val Gln Gly Thr Val Val Leu Asp Gly Asp Glu Tyr Met Gln 149 aaa cgcccg att ggc ccg ctg ctg gct acc ctg ggc cag aac ggc atc 534 Lys Arg ProIle Gly Pro Leu Leu Ala Thr Leu Gly Gln Asn Gly Ile 165 cag gtc gac agcccc acc ggt tgc cca ccg gtc acc gtg cac ggc atg 582 Gln Val Asp Ser ProThr Gly Cys Pro Pro Val Thr Val His Gly Met 181 ggc aag gtc cag gcc aagcgt ttc gag att gat ggt ggt ttg tcc agc 630 Gly Lys Val Gln Ala Lys ArgPhe Glu Ile Asp Gly Gly Leu Ser Ser 197 cag tac gta tcg gcc ctg ctg atgctc gcg gcg tgc ggc gaa gcg ccg 678 Gln Tyr Val Ser Ala Leu Leu Met LeuAla Ala Cys Gly Glu Ala Pro 213 att gaa gtg gcg ctg acc ggc aag gat atcggt gcc cgt ggc tac gtg 726 Ile Glu Val Ala Leu Thr Gly Lys Asp Ile GlyAla Arg Gly Tyr Val 229 gac ctg acc ctc gac tgc atg cgt gcc ttc ggg gcccag gtg gac gcc 774 Asp Leu Thr Leu Asp Cys Met Arg Ala Phe Gly Ala GlnVal Asp Ala 245 gtg gac gac acc acc tgg cgc gtc gcc ccc acc ggc tat accgcc cat 822 Val Asp Asp Thr Thr Trp Arg Val Ala Pro Thr Gly Tyr Thr AlaHis 261 gat tac ctg atc gaa ccc gat gcg tcc gcc gcc acg tat ttg tgg gcc870 Asp Tyr Leu Ile Glu Pro Asp Ala Ser Ala Ala Thr Tyr Leu Trp Ala 277gca gaa gtg ctg acc ggt ggg cgt atc gac atc ggc gta gcc gcg cag 918 AlaGlu Val Leu Thr Gly Gly Arg Ile Asp Ile Gly Val Ala Ala Gln 293 gac ttcacc cag ccc gac gcc aag gcc cag gcc gtg att gcg cag ttc 966 Asp Phe ThrGln Pro Asp Ala Lys Ala Gln Ala Val Ile Ala Gln Phe 309 ccg aac atg caagcc acg gtg gta ggc tcg caa atg cag gat gcg atc 1014 Pro Asn Met Gln AlaThr Val Val Gly Ser Gln Met Gln Asp Ala Ile 325 ccg acc ctg gcg gtg ctcgcc gcg ttc aac aac acc ccg gtg cgt ttc 1062 Pro Thr Leu Ala Val Leu AlaAla Phe Asn Asn Thr Pro Val Arg Phe 341 act gaa ctg gcg aac ctg cgc gtcaag gaa tgt gac cgc gtg cag gcg 1110 Thr Glu Leu Ala Asn Leu Arg Val LysGlu Cys Asp Arg Val Gln Ala 357 ctg cac gat ggc ctc aac gaa att cgc ccgggc ctg gcg acc atc gag 1158 Leu His Asp Gly Leu Asn Glu Ile Arg Pro GlyLeu Ala Thr Ile Glu 373 ggc gat gac ctg ctg gtc gcc agc gac ccg gcc ctggca ggc acc gcc 1206 Gly Asp Asp Leu Leu Val Ala Ser Asp Pro Ala Leu AlaGly Thr Ala 389 tgc acc gca ctg atc gac acc cac gcc gac cat cgc atc gccatg tgc 1254 Cys Thr Ala Leu Ile Asp Thr His Ala Asp His Arg Ile Ala MetCys 405 ttt gcc ctg gcc ggg ctt aaa gtc tcg ggc att cgc att caa gac ccg1302 Phe Ala Leu Ala Gly Leu Lys Val Ser Gly Ile Arg Ile Gln Asp Pro 421gac tgc gtg gcc aag acc tac cct gac tac tgg aaa gcc tgg ccc agc 1350 AspCys Val Ala Lys Thr Tyr Pro Asp Tyr Trp Lys Ala Trp Pro Ser 437 ctg ggcgtt cac cta aac gac tgacacacaa aacctgtagc agagcttgct 1401 Leu Gly ValHis Leu Asn Asp 444 (SEQ ID NO:2) cgcgaaaaac gcacacgtgc cgcgtttgttcaggaaacac gcgttatcgt tgacgtttat 1461 cgagctaagc tcgctcctac attttgcagcgagatcttg 1500 (SEQ ID NO:1)The open reading frame (“ORF;” SEQ ID NO:3) includes bp 40 to 1371 ofSEQ ID NO:1.

Accordingly, the invention features an isolated polypeptide including anamino acid sequence that is at least 62% (i.e., any number between 62%and 100%, e.g., 65, 70, 75, 80, 85, 90, 95, 99 or 100%) identical to SEQID NO:2. When present in a cell, the polypeptide increases the cell'sability to tolerate glyphosate. The polypeptide of the invention can beused for producing an antibody (either monoclonal or polyclonal) thatselectively binds to the polypeptide. The antibody in turn is useful fordetecting the presence of the polypeptide in vivo and in vitro. Forexample, such an antibody can be used to verify the expression of thepolypeptide in a transgenic plant.

An “isolated polypeptide” refers to a polypeptide substantially freefrom naturally associated molecules, i.e., it is at least 10% (i.e., anynumber between 10% and 100%) pure by dry weight. Purity can be measuredby any appropriate standard method, for example, by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul ((1990) Proc. Natl. Acad. Sci. USA87:2264–2268), modified as in Karlin and Altschul ((1993) Proc. Natl.Acad. Sci. USA 90;5873–5877). Such an algorithm is incorporated into theXBLAST programs of Altschul et al. ((1990) J. Mol. Biol. 215:403–410).BLAST protein searches are performed with the XBLAST program, score=50,wordlength=3. Where gaps exist between two sequences, Gapped BLAST isutilized as described in Altschul et al. ((1997) Nucleic Acids Res.25:3389–3402). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST) are used.See www.ncbi.nlm.nih.gov.

The invention further features an isolated nucleic acid characterized inthat it hybridizes under stringent conditions to SEQ ID NO:1 or 3, or acomplementary sequence thereof, as well as a cell (e.g., in a culture orin a transgenic plant) containing a nucleic acid of the invention. Sucha nucleic acid can be at least 15 (e.g., at least 30, 50, 100, 200, 500or 1000) nucleotides in length. An example of a nucleic acid within theinvention is an isolated nucleic acid (e.g., a vector) encoding apolypeptide of the invention, e.g., a nucleic acid that contains SEQ IDNO:1 or 3, where the coding sequence is operably linked to an expressioncontrol sequence. The nucleic acid and the cell can be used forproducing a polypeptide of the invention or generating a transgenicplant. For example, the nucleic acid of the invention can be used todetermine whether an EPSP synthase mRNA is expressed in a cell ortissue. The nucleic acid can also be used as a primer in PCR-baseddetection methods, or as a labeled probe in nucleic acid blots (e.g.,Northern and Southern blots).

A plant cell of this invention can be cultivated to generate atransgenic plant. Such a transgenic plant is within the scope of theinvention. More specifically, the invention features a transgenic plantcomprising an isolated nucleic acid encoding a polypeptide of theinvention that is operably linked to an expression control sequence.Expression of the polypeptide allows the plant to become more tolerantto glyphosate. Examples of such transgenic plants include transgenicsoybean, cotton, alfalfa, canola, flax, tomato, sugar beet, sunflower,potato, tobacco, corn, wheat, rice, lettuce and rape.

An “isolated nucleic acid” is a nucleic acid removed from its naturalenvironment and thus altered “by the hand of man” from its naturalstate. The term therefore covers, for example, (a) a DNA which has thesequence of part of a naturally occurring genomic DNA molecule but isnot flanked by both of the coding sequences that flank that part of themolecule in the genome of the organism in which it naturally occurs; (b)a nucleic acid incorporated into a vector or into the genomic DNA of aprokaryote or eukaryote in a manner such that the resulting molecule isnot identical to any naturally occurring vector or genomic DNA; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), or a restriction fragment;and (d) a recombinant nucleotide sequence that is part of a hybrid gene,i.e., a gene encoding a fusion protein.

By hybridization under “stringent conditions” is meant (1) hybridizationand wash under low ionic strength and high temperature, e.g., in 0.2×SSCand 0.1% SDS at 60° C.; (2) hybridization in the presence of adenaturing agent, e.g., 50% v/v formamide and 0.1% calf serum/0.1%Ficoll at 42° C.; or (3) hybridization between two strands that are atleast 90%, and preferably at least 95%, identical.

The term “operably linked” refers to functional linkage between anexpression control sequence and a nucleic acid sequence. The operablylinked expression control sequence regulates transcription of thenucleic acid sequence. Examples of an “expression control sequence”includes a promoter, a transcription termination signal, an enhancer, asilencer, an insulator, and the like.

In addition, the invention features a method of producing a polypeptideby cultivating a cell containing a nucleic acid encoding a polypeptideof the invention such that the polypeptide is expressed in the cell.Conditions for polypeptide expression are well known in the art. See,e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

The invention also provides a method of increasing a plant's ability totolerate glyphosate. The method involves introducing a vector of theinvention into a plant cell, cultivating the plant cell, andregenerating a transgenic plant from the cell. A transgenic plant thusproduced has increased tolerance to glyphosate compared to a wild-typeplant.

Plants with increased glyphosate tolerance risk less damages whenexposed to the herbicide. Consequently, the invention features a methodof selectively controlling weeds in a field. The method includes growinga transgenic plant of the invention in a field and applying to the fielda sufficient amount of glyphosate. A “sufficient amount” refers to thequantity of the herbicide that enables control of the weeds, yet in themeantime, does not significantly affect the plant, e.g., in quality andyield.

The above-mentioned and other features of this invention and the mannerof obtaining and using them will become more apparent, and will be bestunderstood, by reference to the following description.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a novel EPSP synthase. Unexpectedly, thisenzyme has been found to be highly resistant to glyphosate. For example,bacteria transformed with DNA encoding this enzyme grew in mediacontaining 10–150 mM glyphosate, and a transgenic tobacco grew in thepresence of 15 mM glycosate (see Examples 1 and 5 below).

At the amino acid sequence level, SEQ ID NO:2 has a low homology toknown Class I EPSP synthases (from bacteria such as E. coli andSalmonella typhimurium). For example, the homology between SEQ ID NO:2and the E. coli EPSP synthase is 30.4%, and the homology between SEQ IDNO:2 and the Salmonella typhimurium EPSP synthase is 31.7%. SEQ ID NO:2shows 24.53% homology to Agrobacterial CP4 EPSP synthase disclosed inMonsanto's patents. Moreover, SEQ ID NO:2 does not contain theL-G-N-A-A-T-A (SEQ ID NO:4) sequence at positions corresponding to thea.a. 80–120 region disclosed in Monsanto's patents (e.g., U.S. Pat. No.4,971,908). It also differs from the A-L-L-M-X1-A-P-L-T (SEQ ID NO:5)sequence at positions corresponding to the a.a. 170–210 region disclosedin Monsanto's patents (e.g., U.S. Pat. No. 5,866,775), wherein X1 isalanine, serine or threonine. For example, SEQ ID NO:2 is only 36.6%homologous to a petunia EPSP synthase in the a.a. 170–210 region.Comparison with all known EPSP synthases revealed that SEQ ID NO: 2 hasthe highest (60.7%) similarity to that of EPSP synthase fromAgrobacterium tumefaciens C58 and is 20.53% similar to that of EPSPsynthase from Pseudonomas mallei of the same genus.

Polypeptides

One object of the invention is to provide an isolated polypeptidecomprising an amino acid sequence that is at least 62% (i.e., any numberbetween 62% and 100%, e.g., 65, 70, 75, 80, 85, 90, 95, 99 or 100%)identical to SEQ ID NO:2. A cell expressing this polypeptide becomesmore tolerant to glyphosate (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150 mM glyphosate). Such a polypeptide may be afragment, e.g., a biologically active portion of SEQ ID NO:2, for use asan immunogen or antigen to raise or test (or more generally to bind) ananti-EPSP synthase antibody. In a preferred embodiment, the polypeptideof the invention has an amino acid sequence shown in SEQ ID NO:2. Thepolypeptide of the invention can be isolated from cells (e.g., bacterialcells transformed with a nucleic acid encoding SEQ ID NO:2 as describedExamples 1 and 4) or tissue sources (e.g., tissues from transgenicplants as described in the Example 5) using standard proteinpurification techniques. It can also be produced by recombinant DNAtechniques or synthesized chemically.

Other embodiments include a polypeptide that contains one or morechanges in amino acid sequence, e.g., a change in an amino acid residuewhich is not essential for EPSP synthase's ability to tolerateglyphosate. Such a polypeptide differs in amino acid sequence from SEQID NO:2, yet retains its biological activity. In some embodiments, thedifference is a conservative substitution, while in others, thedifference is a non-conservative substitution. Like point mutations,insertion and deletion mutants can be generated by mutagenesis accordingto the procedures well known in the art.

The term “conservative substitution,” as used herein, denotes thereplacement of an amino acid by another biologically similar residue.Examples of conservative substitutions include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic acid for asparticacid, or glutamine for asparagine, and the like.

The amino acid sequences and structures of EPSP synthases from variousspecies have been well studies. See, e.g., Park et al. (2004) MolecularMicrobiology 51(4):963–971; Eschenburg et al. (2002) Planta 216:129–135;Schonbrunn et al. (2001) PNAS 98 (4):1376–1380; U.S. Pat. No. 4,769,061;U.S. Pat. No. 4,940,835; U.S. Pat. No. 4,971,908; U.S. Pat. No.5,094,945; U.S. Pat. No. 5,310,667; U.S. Pat. No. 5,633,435; U.S. Pat.No. 5,866,775; U.S. Pat. No. 5,145,783; EP 0293358 A2; EP 0409815 A1; WO91/04323; and WO 92/06201.

Comparing with the previously known sequences for EPSP synthases, insome embodiments, a polypeptide of the invention does not contain someof the sequence characteristics described in the above-mentionedpatents. For example, in one embodiment, the polypeptide does notcontain a sequence of L-G-N-A-A-T-A (SEQ ID NO:4) at positionscorresponding to a.a. 119–125 of SEQ ID NO:2.

In another embodiment, the polypeptide does not contain a sequence ofE-R-P-I-X2-X3-L-V-X4-X5-X6-X7-X8-X9-A (SEQ ID NO:6) at positionscorresponding to a.a. 150–164 of SEQ ID NO:2, in wherein X2–5 and X7–8are any amino acid residues, X6 is either arginine or lysine, and X9 iseither aspartic acid or asparagines.

In another embodiment, the polypeptide does not contain a sequence ofR-X10-H-X11-E (SEQ ID NO:7), in which X10 is G, S, T, C, Y, N, Q, D orE; X11 is S or T.

In another embodiment, the polypeptide does not contain a sequence ofG-D-K-X12 (SEQ ID NO:8), in which X12 is S or T.

In another embodiment, the polypeptide does not contain a sequence ofS-A-Q-X13-K (SEQ ID NO:9), in which X13 is A, R, N, D, C, Q, E, G, H, I,L, K, M, F, P, S, T, W, Y or V.

In another embodiment, the polypeptide does not contain a sequence ofN-X14-T-R (SEQ ID NO:10), in which X14 is A, R, N, D, C, Q, E, G, H, I,L, K, M, F, P, S, T, W, Y or V.

In still another embodiment, the polypeptide does not contain a sequenceof A-L-L-M-X15-A-P-L-T (SEQ ID NO:11) at positions corresponding to a.a.202–210 of SEQ ID NO:2, wherein X15 is either alanine, serine orthreonine; or it differs by at least 64% (i.e., any number between 64%and 100%) from other EPSP synthases in this region.

In some embodiments, a polypeptide of the invention contains somesequence characteristics that haven't been found in any other EPSPsynthases. For example, in one embodiment, the polypeptide contains asequence of GKVAPPGSKSITNRALLLAALAKGTSRLSGAL (SEQ ID NO:12) at positionscorresponding to a.a. 44–75 of SEQ ID NO:2.

In another embodiment, the polypeptide contains a sequence ofLFLGNAGTAMRFLTAAVAT (SEQ ID NO:13) at positions corresponding to a.a.117–135 of SEQ ID NO:2.

In another embodiment, the polypeptide contains a sequence ofVLDGDEYMQKRPIGPLLATLGQNGIQV (SEQ ID NO:14) at positions corresponding toa.a. 141–167 of SEQ ID NO:2.

In another embodiment, the polypeptide contains a sequence ofLAVLAAFNNTPVRFTELANLRVKECDRVQALHDGLNEIRPGLATIEGDDLL (SEQ ID NO:15) atpositions corresponding to a.a. 328–378 of SEQ ID NO:2.

In another embodiment, the polypeptide contains a sequence ofIDTHADHRIAMCFALAGL (SEQ ID NO:16) at positions corresponding to a.a.394–411 of SEQ ID NO:2.

In yet another embodiment, the polypeptide contains a sequence ofLTGKDIGARGYVDLTLDC (SEQ ID NO:17) at positions corresponding to a.a.218–235 of SEQ ID NO:2.

In some embodiments, one or more of the underlined amino acids in SEQ IDNOs:12–17 are reserved while other amino acids may be changed.

Furthermore, comparison of SEQ ID NO:2 with E. coli and petunia EPSPsynthases revealed some conserved amino acid residues (underlinedbelow). In some enbodiments, a polypeptide of the invention contains oneor more of these residues (i.e., R151. S196, S197, Q198, D323, N346,K350, R354, R401, and K426 of SEQ ID NO:2).

191                 213 E. coli EPSP synthase (112)DIVLTGEPRMKERPIGHLVDALR (SEQ ID NO:18) SEQ ID NO:2 (139)TVVLDGDEYMQKRPIGPLLATLG (SEQ ID NO:19) Petunia EPSP synthase (191)RYVLDGVPRMRERPISDLVDGLK (SEQ ID NO:20) Consensus (191) IVLDGDPRMKERPIG LVDALK (SEQ ID NO:21) 244          259 E. coli EPSPsynthase (163) DVDGSVSSQFLTALLM (SEQ ID NO:22) SEQ ID NO:2 (190)EIDGGLSSQYVSALLM (SEQ ID NO:23) Petunia EPSP synthase (244)KLSGSISSQYLTALLM (SEQ ID NO:24) Consensus (244) DIDGSISSQYLTALLM (SEQ IDNO:25) 400        413 E. coli EPSP synthase (309) NHIPDAAMTIATAA (SEQ IDNO:26) SEQ ID NO:2 (320) -QMQDAIPTLAVLA (SEQ ID NO:27) Petunia EPSPsynthase (399) NKMPDVAMTLAVVA (SEQ ID NO:28) Consensus (400)N MPDAAMTLAVLA (SEQ ID NO:29) 425         439 E. coli EPSP synthase(334) IYNWRVKETDRLFAM (SEQ ID NO:30) SEQ ID NO:2 (344) LANLRVKECDRVQAL(SEQ ID NO:31) Petunia EPSP synthase (424) VASWRVKETERMIAI (SEQ IDNO:32) Consensus (425) IANWRVKETDRL AI (SEQ ID NO:33) 477       489 E.coli EPSP synthase (381) TYNDHRMAMCFSL (SEQ ID NO:34) SEQ ID NO:2 (396)THADHRIAMCFAL (SEQ ID NO:35) Petunia EPSP synthase (471) TYDDHRMAMAFSL(SEQ ID NO:36) Consensus (477) TY DHRMAMCFSL (SEQ ID NO:37) 503      514E. coli EPSP synthase (407) KCTAKTFPDYFE (SEQ ID NO:38) SEQ ID NO:2(422) DCVAKTYPDYWK (SEQ ID NO:39) Petunia EPSP synthase (497)GCTRKTFPNYFD (SEQ ID NO:40) Consensus (503)  CTAKTFPDYFD (SEQ ID NO:41)

Two or more of the individual embodiments described above may becombined.

In another aspect, the invention provides chimeric or fusion proteinscontaining the polypeptide of the invention. As used herein, a “chimericprotein” or “fusion protein” includes a polypeptide of the inventionlinked to a foreign polypeptide. A “foreign polypeptide” is notsubstantially homologous to a polypeptide of the invention. The foreignpolypeptide can be fused to the N-terminus or C-terminus of thepolypeptide of the invention.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST fusion protein inwhich a polypeptide of the invention is fused to the C-terminus of GST.Such fusion proteins can facilitate the purification of the polypeptide.Alternatively, the fusion protein can contain a heterologous signalsequence at its N-terminus. In certain host cells, expression, secretionor transport of a protein can be increased through use of a heterologoussignal sequence. For example, in a plant cell, a polypeptide of theinvention may be fused with a chloroplast transit peptide. Thechloroplast transit peptide allows the polypeptide to be transportedfrom the cytoplasm of the plant cell into the chloroplast, therebyconferring a substantial degree of glyphosate resistance. Expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of theinvention can be cloned into such an expression vector so that thefusion moiety is linked in-frame to the polypeptide.

Antibodies

In another aspect, the invention provides an antibody against thepolypeptide of the invention, or a fragment thereof (e.g., anantigen-binding fragment thereof). The term “antibody,” as used herein,refers to an immunoglobulin molecule or immunologically active portionthereof, i.e., an antigen-binding portion. It comprises at least one,and preferably two, heavy (H) chain variable regions (abbreviated hereinas VH), and at least one and preferably two light (L) chain variableregions (abbreviated herein as VL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). The extent of the frameworkregion and CDR's has been precisely defined (see, e.g., Kabat et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91–3242, and Chothia et al. (1987) J. Mol. Biol. 196:901–917). Each VHand VL is composed of three CDR's and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

An antibody of the invention can further include a heavy and light chainconstant region, to thereby form a heavy and light immunoglobulin chain,respectively. In one embodiment, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are inter-connected by, e.g.,disulfide bonds. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. The light chain constant region is comprisedof one domain, CL. The variable region of the heavy and light chainscontains a binding domain that interacts with an antigen. The constantregions of the antibodies typically mediate the binding of the antibodyto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (Clq) of the classicalcomplement system.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. Full-length immunoglobulin “light chains” (about 25 KDa or 214amino acids) are encoded by a variable region gene at the N-terminus(about 110 amino acids) and a kappa or lambda constant region gene atthe C-terminus. Full-length immunoglobulin “heavy chains” (about 50 KDaor 446 amino acids), are similarly encoded by a variable region gene(about 116 amino acids) and one of the other aforementioned constantregion genes, e.g., gamma (encoding about 330 amino acids).

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion” or “fragment”), as used herein, refers to one or more fragmentsof a full-length antibody that retain the ability to specifically bindto the antigen, i.e., a polypeptide of the invention. Examples ofantigen-binding fragments of the antibody include, but are not limitedto: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH,CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al. (1989) Nature 341:544–546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); see,e.g., Bird et al. (1988) Science 242:423–426, and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879–5883). Such single chain antibodiesare also encompassed within the term “antigen-binding fragment” of anantibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An antibody of the invention can be a polyclonal or a monoclonalantibody. In other embodiments, the antibody can be recombinantlyproduced, e.g., produced by phage display or by combinatorial methods.Phage display and combinatorial methods for generating antibodies areknown in the art (as described in, e.g., U.S. Pat. No. 6,756,196; U.S.Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370–1372; Hay et al. (1992) Hum Antibod Hybridomas3:81–85; Huse et al. (1989) Science 246:1275–1281; Griffths et al.(1993) EMBO J. 12:725–734; Hawkins et al. (1992) J Mol Biol 226:889–896;Clackson et al. (1991) Nature 352:624–628; Gram et al. (1992) PNAS89:3576–3580; Garrad et al. (1991) Bio/Technology 9:1373–1377;Hoogenboom et al. (1991) Nuc Acid Res 19:4133–4137; and Barbas et al.(1991) PNAS 88:7978–7982).

In preferred embodiments, an antibody can be made by immunizing with apurified antigen, crude tissue preparation, whole cell, lysed cell, orcell fraction.

A full-length polypeptide of the invention or an antigenic peptidefragment of it can be used as an immunogen or can be used to identifyantibodies made with other immunogens, e.g., cells, and the like. Theantigenic peptide should include at least 8 amino acid residues of theamino acid sequence shown in SEQ ID NO:2 and encompasses an epitope.Preferably, the antigenic peptide includes at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Antibodies which bind only the native form of the polypeptide of theinvention, only the denatured or otherwise non-native form, or whichbind both, are with in the invention. Antibodies with linear orconformational epitopes are within the invention. Conformationalepitopes can sometimes be identified by identifying antibodies whichbind to native but not denatured form of the polypeptide.

Preferred epitopes encompassed by the antigenic peptide are regionslocated on the surface of the polypeptide, e.g., hydrophilic regions, aswell as regions with high antigenicity. For example, an Emini surfaceprobability analysis of the polypeptide sequence can be used to indicatethe regions that have a particularly high probability of being localizedto the surface of the polypeptide and are thus likely to constitutesurface residues useful for targeting antibody production.

In preferred embodiments, antibodies can bind one or more of purifiedantigens, tissues, e.g., tissue sections, whole cells, lysed cells, andcell fractions.

The antibody can be a single chain antibody. A single-chain antibody(scFV) may be engineered (see, for example, Colcher et al. (1999) Ann NY Acad Sci 880:263–80 and Reiter (1996) Clin Cancer Res 2:245–52). Thesingle chain antibody can be dimerized or multimerized to generatemultivalent antibodies having specificities for different epitopes ofthe same target.

An antibody (e.g., monoclonal antibody) can be used to isolate apolypeptide of the invention by standard techniques, such as affinitychromatography or immunoprecipitation. Moreover, an antibody can be usedto detect the polypeptide (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the polypeptide. Detection can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance (i.e.,antibody labelling). Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase and acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;examples of luminescent materials include luminol; examples ofbioluminescent materials include luciferase, luciferin and aequorin, andexamples of suitable radioactive materials include ¹²⁵I, ¹³¹I, ³⁵S or³H.

The invention also includes a nucleic acid which encodes an antibody ofthe invention. Also included are a vector containing the nucleic acidand a cell transformed with the vector, particularly a cell which isuseful for producing an antibody. The invention also includes a methodof using a cell, e.g., a hybridoma, to make an antibody of theinvention.

Nucleic Acids

In yet another aspect, the invention provides an isolated nucleic acidthat encodes a polypeptide of the invention. Also included is a nucleicacid fragment suitable for use as a hybridization probe, which can beused, e.g., to identify a nucleic acid (a DNA or mRNA) encoding apolypeptide of the invention, and an fragment suitable for use as aprimer, e.g., a PCR primer for amplification or mutation of the nucleicacid.

In one embodiment, an isolated nucleic acid of the invention includesthe nucleotide sequence shown in SEQ ID NO:1 or a portion thereof. Forexample, the nucleic acid may include the coding region of SEQ ID NO:1,as shown in SEQ ID NO:3. In another embodiment, the isolated nucleicacid of the invention includes a nucleic acid molecule which is acomplement of the nucleotide sequence shown in SEQ ID NO:1 or 3, or aportion thereof. In other embodiments, the nucleic acid of the inventionis sufficiently complementary to the nucleotide sequence shown in SEQ IDNO:1 or 3, such that it can hybridize (e.g., under stringency conditionsdescribed above) to the nucleotide sequence shown in SEQ ID NO:1 or 3,thereby forming a stable duplex.

A nucleic acid of the invention can include only a portion of thenucleic acid sequence of SEQ ID NO:1 or 3. For example, such a nucleicacid can include a fragment which can be used as a probe or primer or afragment encoding a portion of a polypeptide of the invention, e.g., animmunogenic or biologically active portion of the polypeptide. Thenucleotide sequence determined from the cloning of the new EPSP synthasegene allows for the generation of probes and primers designed for use inidentifying or cloning other EPSP synthases, or fragments thereof, aswell as EPSP synthase homologues, or fragments thereof, from otherspecies.

Typically a probe/primer is an isolated or purified oligonucleotide. Theoligonucleotide typically includes a region of nucleotide sequence thathybridizes under stringency conditions described above to at least about7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35,40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense orantisense sequence of SEQ ID NO:1 or 3, or of a naturally occurringallelic variant or mutant of SEQ ID NO:1 or 3. Preferably, a primer isless than about 200, 150, 120, 100, 50, 30, 20 or 10 nucleotides inlength. A probe may be 10, 20, 30, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 or more nucleotides in length. In one embodiment,the probe or primer is attached to a solid support, e.g., in amicroarray analysis.

The invention further encompasses nucleic acids that differ from thenucleotide sequence shown in SEQ ID NO:1 or 3. Such differences can bedue to degeneracy of the genetic code (and result in a nucleic acidwhich encodes the same polypeptide). In another embodiment, an isolatednucleic acid of the invention has a nucleotide sequence encoding apolypeptide having an amino acid sequence which differs, by at least 1,but less than 5, 10, 20, 50, 100 or 150 amino acid residues that shownin SEQ ID NO:2. If alignment is needed for this comparison, thesequences should be aligned for maximum homology. “Looped” out sequencesfrom deletions or insertions, or mismatches, are considered differences.

In one embodiment, a nucleic acid includes a nucleotide sequence thatincludes part, or all, of the coding region and extends into either (orboth) the 5′ or 3′ non-coding region.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non-preferred, for a particular expression system. Forexample, the nucleic acid can be one in which at least one codon, andpreferably at least 10% or 20% of the codons, has been altered such thatthe sequence is optimized for expression e.g, in bacterial or plantcells.

Variants of the nucleic acid of the invention can be naturallyoccurring, such as allelic variants (same locus), homologs (differentlocus), and orthologs (different organism) or can be non-naturallyoccurring. Non-naturally occurring variants can be made by mutagenesistechniques, including those applied to polynucleotides, cells, ororganisms. The variants can contain nucleotide substitutions, deletions,inversions and insertions. Variation can occur in either or both thecoding and non-coding regions. The variations can produce bothconservative and non-conservative amino acid substitutions describedabove.

Allelic variants include both functional and non-functional proteins.Functional allelic variants are naturally occurring amino acid sequencevariants within a population that maintain the EPSP synthase activity.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:2, or substitution,deletion or insertion of non-critical residues in non-critical regionsof the polypeptide. Non-functional allelic variants arenaturally-occurring amino acid sequence variants that do not have theEPSP synthase activity. Non-functional allelic variants will typicallycontain a non-conservative substitution, a deletion, or insertion, orpremature truncation of the amino acid sequence of SEQ ID NO:2, or asubstitution, insertion, or deletion in critical residues or criticalregions of the protein.

Vectors and Cultured Cells

An additional aspect of the invention includes vectors containing anucleic acid encoding a polypeptide of the invention. Examples of suchvectors are described in Examples 1 and 4 below. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked and can include aplasmid or cosmid vector. The vector can be capable of autonomousreplication or it can integrate into a host DNA.

A vector can include a nucleic acid of the invention in a form suitablefor expression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more expression controlsequences operatively linked to the nucleic acid sequence to beexpressed. Expression control sequences include those which directconstitutive expression of a nucleotide sequence, as well astissue-specific regulatory and/or inducible sequences. The design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce the polypeptide, as well as a fusionpolypeptide, a mutant form of the polypeptide, and the like.

The recombinant expression vectors of the invention can be designed forexpression of the polypeptide of the invention in prokaryotic oreukaryotic cells. For example, polypeptides of the invention can beexpressed in E. coli or plant cells. Suitable host cells are discussedfurther in Goeddel (1990) Gene Expression Technology, Methods inEnzymology 185, Academic Press, San Diego, Calif. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example, using T7 promoter regulatory sequences and T7polymerase.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion polypeptides.Fusion vectors add a number of amino acids to a polypeptide encodedtherein, usually to the amino terminus of the recombinant polypeptide.Such fusion vectors typically serve three purposes: (1) to increaseexpression of a recombinant polypeptide; (2) to increase the solubilityof the recombinant polypeptide; and (3) to aid in the purification ofthe recombinant polypeptide by acting as a ligand in affinitypurification. Often, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant polypeptide to enableseparation of the recombinant polypeptide from the fusion moietysubsequent to purification of the fusion polyprptide. Such enzymes, andtheir cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Typical fusion expression vectors include pGEX (PharmaciaBiotech, Inc.; Smith and Johnson (1988) Gene 67:31–40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant polypeptide.Purified fusion polypeptides can be used in EPSP synthase activityassays or to generate antibodies.

To maximize recombinant polypeptide expression in E. coli, thepolypeptide is usually expressed in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant polypeptide(Gottesman (1990) Gene Expression Technology, Methods in Enzymology 185,Academic Press, San Diego, Calif., p119–128). Another strategy is toalter the nucleic acid sequence of the nucleic acid to be inserted intoan expression vector so that the individual codons for each amino acidare those preferentially utilized in E. coli (Wada et al. (1992) NucleicAcids Res. 20:2111–2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the promoter is an inducible promoter, e.g., apromoter regulated by a heterologous polypeptide (e.g., thetetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g.,Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA89:5547).

When used in plant cells, promoters which are known or found to causetranscription of EPSP synthase genes in plant cells can be used (see,e.g., U.S. Pat. No. 4,940,835). Such promoters may be obtained fromplants or viruses and include, but are not limited to, the 35S and 19Spromoters of cauliflower mosaic virus and promoters isolated from plantgenes such as EPSP synthases and ssRUBISCO genes, and promoters obtainedfrom T-DNA genes of Agrobacterium tumefaciens such as nopaline andmannopine synthases genes.

These promoters may be further modified if desired to alter theirexpression characteristics. For example, the CaMV 35S promoter may beligated to the portion of the ssRUBISCO gene which represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The RNA produced from theEPSP synthase gene may contain a 5′ non-translated leader sequence. Thissequence may be derived from any gene and may be specifically modifiedso as to increase translation of the mRNA. The 5′ non-translated regionsmay be derived from viral RNAs, other suitable eukaryotic genes or asynthetic gene sequence. It may be part of the 5′ end of thenon-translated region of the coding sequence for the EPSP synthasepolypeptide or derived from an unrelated promoter or coding sequence.

In one embodiment, a polypeptide of the invention is fused to achloroplast transit peptide (CTP). After the fusion polypeptide isproduced in the cytoplasm of a transformed plant cell, the CTP leadersequence causes the polypeptide to be imported into chloroplasts, andthe CTP leader sequence is removed from the remainder of the polypeptideso that an active portion of the polypeptide exists and functions insidethe chloroplast. Suitable CTP's may be obtained from various sources.Most preferably, the CTP is obtained from the endogenous EPSP synthasegene of the subject plant to be transformed. Alternately, a CTP from anEPSP synthase gene of another plant or a CTP from another plant gene maybe used. Suitable CTP sequences can be determined, e.g., by assaying thechloroplast uptake of the polypeptide comprising the CTP of interest asdescribed in U.S. Pat. No. 4,940,835.

The 3′ non-translated region may contain a polyadenylation signal whichfunctions in plants to cause the addition of polyadenylate nucleotidesto the 3′ end of the mRNA. The 3′ non-translated region naturallyassociated with a plant EPSP synthase gene may be used. Examples ofother suitable 3′ regions are the 3′ transcribed, non-translated regionscontaining the polyadenylation signal of the nopaline synthase (NOS)gene of the Agrobacterium tumor-inducing (Ti) plasmid or the conglycinin(7S) storage protein gene.

Another aspect the invention provides a cultured cell which includes anucleic acid of the invention, e.g., within a recombinant expressionvector which allows it to homologously recombine into a specific site ofthe host cell's genome. A host cell can be any prokaryotic or eukaryoticcell. For example, a polypeptide of the invention can be expressed in abacterial cell or a plant cell. Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation.

Tansgenic Plants

A nucleic acid encoding a polypeptide of the invention is inserted intothe genome of a plant by any suitable method known in the art. Suitableplants include, but are not limited to, soybean, cotton, alfalfa,canola, flax, tomato, sugar beet, sunflower, potato, tobacco, corn,wheat, rice, lettuce and rape.

Suitable plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens as well as those described in, e.g.Herrera-Estrella et al. (1983) Nature 303:209, Bevan et al. (1983)Nature 304:184, and EPO publication 120,516). In addition to planttransformation vectors derived from the Ti or rootinducing (Ri) plasmidsof Agrobacterium, alternative methods can be used to insert the nucleicacid into plant cells. Such methods may involve, for example, liposomes,electroporation, chemicals which increase free DNA uptake, and the useof viruses or pollen as vectors. If desired, more than one copy of thenucleic acid may be inserted into the chromosomes of a plant, by methodssuch as repeating the transformation and selection cycle more than once.

Transformed plant cells can be regenerated into differentiated plantsusing standard nutrient media supplemented with selected shoot-inducingor root-inducing hormones, using methods described in WO 084/02920 orother methods known to those skilled in the art. For example, glyphosatetolerant transgenic tobacco can be generated using the materials andmethods described in Examples 3–5 below.

Uses

A cultured cell or transgenic plant of the invention can be used toproduce (i.e., express) a polypeptide of the invention. Accordingly, theinvention further provides a method for producing a polypeptide of theinvention using a cultured cell or transgenic plant described above. Inone embodiment, the method includes culturing the cell or transgenicplant (into which a recombinant expression vector encoding a polypeptideof the invention has been introduced) in a suitable medium such that thepolypeptide is produced. In another embodiment, the method furtherincludes isolating the polypeptide from the medium, the cultured cell orthe transgenic plant.

A nucleic acid encoding a polypeptide of the invention fused to a CTPsequence also provides a useful selectable marker for plant celltransformation, when transformed and untransformed cells are contactedwith appropriate concentrations of glyphosate (which can be routinelydetermined for any type of plant). The conferrable trait of glyphosateresistance may be particularly useful with certain types of plants (suchas alfalfa, soybean and other legumes) which do not exhibit clearselectability using other selectable marker genes (such as kanamycin,methotrexate or hygromycin resistance genes).

In another aspect, the invention provides a method for improving theability of a plant (e.g., a crop) to tolerate glyphosate by introducinga nucleic acid encoding a polypeptide of the invention (e.g., a vectorof the invention) into a plant cell. The transformed cell is cultivatedand used to generate a transgenic plant. Expression of the polypeptidein the plant confers higher glyphosate tolerance to the plant.

In yet another aspect, the invention provides a method for selectivelycontrolling weeds in a field where a transgenic plant (e.g., a crop) ofthe invention is grown. As the transgenic plant is more tolerant toglyphosate, the herbicide may be applied to the field in an amountsufficient to selectively kill or control weeds that may also be growingin the field that are not glyphosate tolerant. This allows the desiredglyphosate tolerant plant to take full advantage of the availablenutrients in the field for an improved quality and yield.

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

EXAMPLES Example 1

Cloning of High Glyphosate Tolerance DNA Fragment

1. Sample Collection from Soil Highly Contaminated with Glyphosate

There are many different bacterial strains which are highly tolerant toglyphosate or other herbicides in the natural environment, especially inthose soils sprayed with glyphosate for a long time. Samples werecollected from the soil in the open packaging area in Hebei QifengChemical Industrial Co., Ltd. The soil was contaminated with about 50%glyphosate for more than 10 years.

2. Isolation of Total DNA at the Community Level from Soil HighlyContaminated with Glyphosate Using Non-Bacterial-Culture Method

Two grams of soil was added into 0.6 g of small beads (diameter <0.11mm), and vortexed at 4000 rpm twice. The mixture was suspended in 300 μllysis buffer containing 2% v/v SDS, 12% v/v equilibrated phenol-Tris.HCl(pH 8.0) on ice for one hour. An equal volume (about 700 μl) ofequilibrated phenol-Tris.HCl (pH 8.0) buffer was added. The sample wasmixed well, followed by centrifuge at 13000 rpm for 5 minutes. Thesupernatant was mixed with 0.1× volume of 3 M NaAc (pH 5.2), andsubsequently 0.6× volume of iso-propylacohol to precipitate DNA. The DNApellet (crude DNA) was dissolved in 200 μl 1×TE buffer. 100 mg CsCl wasweighed and added into a new 1.5 ml Enppendorf tube, and gently mixedwith 100 μl crude DNA. The sample was incubated in the dark for 1–3hours to allow CsCl to dissolve, and then centrifuged at 13000 rpm for20 minutes at room temperature. 400 μl sterile de-ionized water and 300μl iso-propylacohol were added to the supernatant, and the mixture wasincubated at room temperature for 30 minutes. The solution wascentrifuged at 13000 rpm for 20 minutes at room temperature. Thesupernatant was drained off. The pellet was dissolved in 100 μl 1×TE and40 μl 8 M KAc, and incubated for 15 minutes at room temperature. Thesolution was centrifuged at 13000 rpm for 15 minutes at 4° C. Thesupernatant was mixed with 0.6× volume of iso-propylalcohol, incubatedfor 30 minutes, and centrifuged at 15000 rpm for 20 minutes at roomtemperature. The DNA pellet was dissolved in 100 μl 1×TE buffer. TheWizard spin column clean-up kit was used to purify DNA samples. Thepurified DNA was dissolved in 10 mM TE (Tris-EDTA, pH 8.0) to an endvolume of 100 μl.

3. Construction of Community Genomic DNA Cosmid Library

A SuperCos1 Cosmid Vector kit (Stratagene Co., Ltd) was used toconstruct a community genomic DNA cosmid library. The community genomicDNA was digested with restriction endonuclease Sau3AI at 0.006unit/microgram purified DNA. DNA fragments of about 40 kb were collectedusing the freeze-thaw method, and then ligated with SuperCos1/BamH1vector. A cosmid genomic DNA library was constructed according to themanual of Stratagene Co., Ltd. For screening of glyphosate tolerantclones, JM109 E. coli was used as the acceptor bacterium for the genomiclibrary construct, as glyphosate is only effective on an inhibitorymedium such as M9 and MOPS. The ligated DNA was packaged into lambdaphage particles (Stratagene, Gigapack Gold) according to themanufacturer's protocol. E. coli strain JM109 was infected with thepackaged product. The transformants were selected on tetracycline LBmedia. Positive clones were then plated on LB media containingtetracycline and tetracycline+kanamycin, respectively. Clones growing onboth media were considered as negative recombinants resulting fromvector ligated with vector DNA. The number of positive transformants wascalculated as the total number of colonies minus background falsenegative colonies. This number was used to determine the titer of thegenomic library, which was 4×10⁶ pfu/ml, meeting the requirement forlibrary construction (>10⁶ pfu/ml).

4. Screening of Glyphosate Tolerant Transformants

The transformants were plated on MOPS LB media containing phosphorussalt and 10 mM glyphosate. About ten clones grew up two days followinginoculation. These clones were replicated onto MOPS LB media containingphosphorus salt and different concentrations of glyphosate. Thetransformant with highest glyphosate tolerance could grow on mediacontaining 60 mM of glyphosate. The plasmid DNA, pGR1, was extracted andtransformed back into E. coli JM109 by electroporation. As a control,the vector plasmid, pLA2917, was also transformed into JM109. Thetransformants were picked and tested on MOPS plates containing 20 mMglyphosate for glyphosate tolerance. The results showed that all pGR1transformants have the ability to tolerate glyphosate, indicating thatthis ability resulted from transformation of pGR1.

5. Subcloning of High Glyphosate Tolerance DNA Fragment andCharacterization of Glyphosate Tolerance

Plasmid pGR1 DNA that showed highest tolerance to glyphosate wasdigested with different endonucleases: HindIII, PstI, HindIII+PstI,respectively. The inserted fragments were ligated with a vector plasmidDNA (pBlueScript KS or pGEM-3zf), and transformed into E. coli strainJM109. The transformants were selected on L-Broth media containingampicillin by their colors (blue or white). The DNA of white colonieswas extracted using the boiling method and screened for differentrecombinants. The recombinants were screened on MOPS media containing 20mM glyphosate. Among the subclones, one glyphosate tolerant subclone,pGRH1, was chosen. The pGRH1 plasmid DNA was extracted andre-transformed into JM109. The vector plasmid DNA was used as a control.The results showed that all pGRH1 transformants had the ability totolerate glyphosate, indicating that this ability resulted fromtransformation of pGRH1. Based on the restriction enzyme digestionanalysis of pGRH1 plasmid DNA, the full-length of the inserted fragmentis 2.3 kb.

JM109 transformants containing pGR1, pGRH1, and the control vectorplasmid pGEM-3zf were inoculated at the same concentration (1%, based onthe OD (absorbance density) values) into MOPS liquid media containing 50mM and 150 mM glyphosate. The bacterial cultures were incubated at 37°C. for 24 hours with shaking. The ODs were measured at 600 nm. Theresults showed that JM109 recombinants containing pGR1 or pGRH1 couldtolerate 150 mM glyphosate and grow normally, whereas JM109 recombinantscontaining the control vector plasmid could not.

Example 2

Sequence Analysis of High Glyphosate Tolerance Gene and Characterizationof EPSP Synthase Functions

1. Sequence Analysis of High Glyphosate Tolerance Gene

The full-length of the subcloned high glyphosate tolerance DNA fragmentin Example 1 was sequenced. The sequencing results showed that thisfragment, SEQ ID NO:1, is 1500 bp in length with an open reading frameof 1332 bp from bp 40 to 1371 (SEQ ID NO:3), encoding a predicated EPSPsynthase of 444 amino acids (SEQ ID NO:2). A blast search showed thatthe subcloned high glyphosate tolerance DNA fragment shares no identitywith other reported EPSP synthase genes (aroA) at the nucleotide level.

2. Characterization of EPSP Synthase Encoded by High GlyphosateTolerance DNA Fragment

DNA sequence analysis showed that high glyphosate tolerance subclonepGRH1 contains an open reading frame (ORF) for an EPSP synthase. Inorder to verify that the polypeptide encoded by this ORF exhibitsglyphosate tolerance, the EPSP synthase mutant E. coli strain ER2799 wasutilized as a receptor bacterium in a complementation assay. The highglyphosate tolerance subclone pGRH1 plasmid DNA was transformed intoER2799 using the CaCl₂ method. Transformants were picked up with steriletoothpicks and inoculated on MOPS media plates containing glyphosate.ER2799 and the vector plasmid (pBlueScript KS or pGEM-3zf) were used asnegative controls. If the EPSP synthase encoded by pGRH1 complements theE. coli mutation, the transformants can grow by synthesizing amino acidsfrom the inorganic substances in the restricted medium. Otherwise, thetransformants cannot grow.

The complementation assay results revealed that DNA fragments in R3H1and R7H1 transformed with pGRH1 could complement the E. coli EPSPsynthase mutation. Hence, it was confirmed that the DNA fragment insubclone pGRH1 contains a fully functional EPSP synthase gene.

Example 3

Artificial Synthesis of High Glyphosate Tolerance EPSP Synthase Gene

Based on the sequence of the 1332 bp open reading frame in SEQ ID NO:1,the EPSP gene were divided into eight regions. Sixteen strands (positiveand negative strands for each region) were designed and synthesized.These strands were 150–200 bp in length and had sequences for cohesiveends after annealing. The positive strands and their complementarynegative strands were annealed to generate 8 double-stranded fragmentswith cohesive ends. All these fragments were mixed together and ligatedinto a full-length EPSP synthase gene with T4 ligase. This syntheticfragment contains 40–1374 bp of SEQ ID NO:1 and has XbaI and SacIrecognition sites at the ends of the EPSP synthase gene.

This synthetic gene with XbaI and SacI recognition sites at its 5′- and3′-ends was then used in construction of a plant expression vectorcontaining the high glyphosate tolerance EPSP synthase gene.

Example 4

Construction of Plant Expression Vector Containing High GlyphosateTolerance EPSP Synthase Gene

The method used in construction of a plant expression vector containinga high glyphosate tolerance EPSP synthase gene was as follows:

A. The plant expression vector pBI121 (Clontech Co., Ltd) andpCAMBIA2301 (Clontech Co., Ltd) plasmid DNA was digested with twoendonucleases HindIII and EcoRI. The fragment p35S-GUS-Nos-ter from thepBI121 plasmid DNA was ligated with pCAMBIA2301 plasmid DNA, resultingin an intermediate vector p35S-2301-GUS.

B. Plasmid p35S-2301-GUS DNA was digested with two endonucleases XbaIand SacI, and ligated with the artificially synthesized EPSP synthasegene. A plant expression vector containing a high glyphosate toleranceEPSP synthase gene was thus obtained by replacing the GUS gene in theintermediate vector p35S-2301-GUS with the artificially synthesized EPSPsynthase gene. The expression vector was then transformed intoAgrobacterium tumefaciens EHA105, which was subsequently transferredinto model plant tobacco to generate plants highly tolerant toglyphosate.

Example 5

Transformation of Tobacco to Generate Plants Highly Tolerant toGlyphosate Using Leaf Disc Protocol

(1) A positive Agrobacterium tumefaciens EHA105 clone derived fromExample 4 was picked up with a sterilized toothpick and inoculated into2 ml YPE liquid containing kanamycin and streptomycin. The culture wasincubated with shaking at 200 rpm and 28° C. for 24–36 hours.

(2) The culture was centrifuged at 4000 rpm for 15 minutes at roomtemperature.

(3) The upper aqueous layer was discarded and the pellet was resuspendedin ½ MS solution, diluted to 5–20× of the original volume. OD₆₀₀(absorbance density at 600 μm) was about 0.5.

(4) Transformation of tobacco was carried out according to the leaf disctransformation protocol, using sterilized tissue of a healthy leaf ofabout 2 weeks old. The main vein was removed and the leaf was cut intosmall pieces of 2 cm².

(5) The leaf discs were placed into prepared agrobacterial liquid for2–5 minutes. The liquid was then drained off with sterile filters. Theleaf discs were placed upside down on MS media plates and incubated inthe dark at 28° C. for 48 hours.

(6) The discs were transferred, still upside down, to selection plates(MS+6-BA 1.0 mg/l+NAA 0.1 mg/l+Kan 50 mg/l+carbenicillin 250 mg/l). Theplates were incubated at 25–28° C. under the light. After 7–15 days,callus formed.

(7) After about 20 days, shoots were cut from the callus when they werelarge enough to be distinguished from the stems. The shoots were placedinto rooting media (½ MS+NAA 0.5 mg/l+Kanamycin 25 mg/l). Root formationoccurred in 2–7 days.

(8) After development of the roots, plants were taken out. The attachedsolid culture media was washed off with sterile water. The plants wereinitially kept in a high humidity environment such as a plasticcontainer with a glass cover for a few days. After the plants had grownstronger, the glass covers were removed, and the plants were transferredto solid culture media containing 10 mM glyphosate for screening ofglyphosate tolerant plants.

(9) The glyphosate tolerant plants were reconfirmed by Southern,Northern and Westhern analyses.

(10) Glyphosate tolerance assays in greenhouse also confirmed that thetransgenic plants could grow well in media containing 15 mM glyphosate.

While the foregoing has been described in considerable detail and interms of preferred embodiments, these are not to be construed aslimitations on the disclosure or claims to follow. Modifications andchanges that are within the purview of those skilled in the art areintended to fall within the scope of the following claims. Allliteratures cited herein are incorporated by reference in theirentirety.

1. An isolated polypeptide comprising an amino acid sequence that is atleast 95% identical to SEQ ID NO:2, wherein the polypeptide, whenpresent in a cell, increases the cell's ability to tolerate glyphosate.2. The isolated polypeptide of claim 1, wherein the amino acid sequenceis at least 99% identical to SEQ ID NO:
 2. 3. The isolated polypeptideof claim 1 comprising the amino acid sequence of SEQ ID NO:2.
 4. Anisolated polypeptide consisting of the amino acid sequence of SEQ IDNO:2.